The invention relates to a method for the screening of a non-recombinant cell line capable, under appropriate conditions, of selectively expressing levels of a target protein, preferably an isoenzyme of PDE4 A.
The invention also concerns methods for the screening of a candidate molecule that modulates the expression or the activity of human phosphodiesterase 4A.
Cyclic nucleotides are important second messengers that regulate and mediate a number of cellular responses to extra-cellular signals, such as hormones, light and neuro-transmitters.
The cyclic nucleotide phosphodiesterases regulate the cellular concentrations of cyclic nucleotides and thereby play a role in signal transduction. To date, nine different classes of phosphodiesterases have been identified and, among them, phosphodiesterase 4, 7, 8 and 9 have been recognized as cAMP-specific enzymes.
Investigations of recent years revealed that isoenzymes of cyclic-3xe2x80x2-5xe2x80x2-nucleotide phosphodiesterases are critically important components of the cyclic-3xe2x80x2, 5xe2x80x2-adenosine monophosphate (cAMP) protein kinase A (PKA) signalling pathway.
The experimental data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on airway epithelial cells.
The cAMP phosphodiesterases are proteins encoded by 4 genes in mammals (PDE 4A, B, C, D) that encode multiple PDE isoforms generated from alternative spliced mRNA transcripts. However, the physiological significance of this diversity of cAMP specific PDE isoforms is not yet known, although it has been shown that the rat PDE4 genes have different patterns of expression in tissues.
PDE4 isoenzymes are likely to be good targets for molecules acting in diseases caused or regulated by cyclic nucleotide-modulated transduction mechanisms. Pharmaceutical interest in this area has been further sparked by the hypothesis that different PDE isoenzymes having distinct sequences at regulatory and catalytic sites could allow the development of selective therapeutic agents that can target a specific cyclic nucleotide pool in a very limited number of cell types. This in turn holds great promise of being able to limit the toxic effects of many of the first generation selective PDE4 inhibitors.
Regulation of PDE4 gene expression appears to be a complex process. In various cell-based differentiation systems it has been demonstrated that elevation of intracellular cAMP levels plays a pivotal promoting role.
In general, PDE4 activity can be short-term or long-term regulated; short-term regulation of PDE4 activity is a cAMP-dependent protein kinase phosphorylation of the isoenzyme triggered by intracellular cAMP-elevating agents or a direct activation of PDE4 by phosphatidic acid. Long-term up-regulation is also realized by increased intracellular cAMP concentrations but requires both protein and mRNA synthesis. An interesting feature is that different types of regulation can occur in the same cell-type. For example, cells possess a phospholipid-sensitive form of PDE4 which can be activated by anionic phospholipids.
It has been shown that cAMP-increasing agents increased PDE4 activity in this cell-type by enhancement of the transcription of certain PDE4 subtypes (PDE4A and B).
Consequently, there is a great need for new molecules that selectively modulate the expression or the activity of the human PDE 4A, and particularly molecules that are able to inhibit the expression or the activity of the human PDE4A.
The invention concerns a method for the screening of a non-recombinant cell line capable, under appropriate conditions, of exhibiting an upregulated expression of a target protein, preferably of an isoenzyme of PDE4, naturally expressed in the cell. The method comprises:
providing a non-recombinant cell line;
treating the non-recombinant cell line with a concentration of a phorbol ester, preferably PMA, ranging from 0.1 xcexcM to 3 xcexcM, for a period of time ranging from 12 hours to 10 days; preferably from 2 to 7 days, more preferably from 4 to 7 days: and
measuring the amount of target protein produced by the treated non-recombinant cell line and comparing the measured amount with the amount of protein produced under normal culturing conditions by the non-recombinant cell line prior to phorbol ester treatment.
This screening method of the invention exploits the inventor""s findings that a human promyelocytic cell line presents significantly upregulated expression of a specific isoenzyme of phosphodiesterase, namely phosphodiesterase 4A, and a substantially downregulated expression of other PDE4 isoenzymes, preferably unmeasurable levels of all other PDE4 isoenzymes, when treated with a phorbol ester such as PMA.
The present invention also concerns methods for the screening of candidate molecules that modulate the expression or the activity of human phosphodiesterase 4A.
A first screening method of the invention comprises the step of quantifying cAMP content in phorbol ester-treated non-recombinant cells incubated with a candidate molecule to be assayed.
A second screening method of the invention comprises a step wherein an expression analysis of human phosphodiesterase 4A is performed. The candidate molecules selected by the screening methods of the invention are potentially useful as therapeutic molecules for diseases caused or regulated by cyclic nucleotide-modulated transduction mechanisms, airway disorders such as asthma, pulmonary hypertension, COPD (constructive obstructive pulmonary disease), CNS related diseases such as depression and other inflammatory disorders including atopic dermatitis, psoriasis, Crohn""s disease, ulcerative colitis, acute respiratory distress syndrome and rheumatoid arthritis.
The invention also concerns a method for determining the selectivity of a candidate molecule for a PDE4 subtype. The method comprises:
providing a non-recombinant cell line culture presenting a significantly upregulated expression of PDE4A and a substantially downregulated expression of other PDE4 isoenzymes, preferably unmeasurable levels of all other PDE4 isoenzymes;
adding said candidate compound to said cell line culture; and
quantifying the cAMP content in said culture.